Abstract
Background: Human SP-A1 and SP-A2, encoded by SFTPA1 and SFTPA2, and their genetic variants differentially impact alveolar macrophage (AM) functions and regulation, including the miRNome. We investigated whether miRNome differences previously observed between AM from SP-A2 and SP-A1/SP-A2 mice are due to continued qualitative differences or a delayed response of mice carrying a single gene. Methods: Human transgenic (hTG) mice, carrying SP-A2 or both SP-A genes, and SP-A-KO mice were exposed to filtered air (FA) or ozone (O3). AM miRNA levels, target gene expression, and pathways determined 18 h after O3 exposure. RESULTS: We found (a) differences in miRNome due to sex, SP-A genotype, and exposure; (b) miRNome of both sexes was largely downregulated by O3, and co-ex had fewer changed (≥2-fold) miRNAs than either group; (c) the number and direction of the expression of genes with significant changes in males and females in co-ex are almost the opposite of those in SP-A2; (d) the same pathways were found in the studied groups; and (e) O3 exposure attenuated sex differences with a higher number of genotype-dependent and genotype-independent miRNAs common in both sexes after O3 exposure. Conclusion: Qualitative differences between SP-A2 and co-ex persist 18 h post-O3, and O3 attenuates sex differences.
Highlights
Ozone (O3) is a reactive oxidant gas that is a major component of air pollution [1,2]
The expression levels of the following genes were studied by qRT-PCR as described previously [85] in male and female SP-A2, co-ex, and KO alveolar macrophage (AM): Argonaute 2 (AGO2), AKT Serine/Threonine Kinase 1 (AKT1), Arginase 1 (ARG1), B-cell lymphoma 2 (BCL2), Caspase 3 (CASP3), Caspase 8 (CASP8), Caspase 9 (CASP9), Cyclin D1 (CCND1), Cyclin D2 (CCND2), Cyclin E1 (CCNE1), Cyclin-dependent kinase 2 (CDK2), Cyclin-dependent kinase 7 (CDK7), Cyclin dependent kinase inhibitor 2 (CDKN2A), Catenin alpha 1 (CTNNB1), Dead-box helicase 20 (DDX20), E2F transcription factor3 (E2F3), Early growth response 2 (EGR2), Forkhead box O1 (FOXO1), Growth arrest and DNA damage inducible alpha (GADD45A), Interleukin 6 (IL6), Interleukin 10 (IL10), Interleukin 2 receptor subunit gamma (IL2RG), Jun proto-oncogene (JUN), MDTH, Matrix metalloproteinase 9 (MMP9), MYC proto-oncogene (MYC), Myeloid differentiation primary response 88 (MYD88), Peroxisome proliferator activated receptor alpha (PPARA), Phosphatase and tensin homolog (PTEN), SMAD family member 2 (SMAD2), signal transducer and activator of transcription 3 (STAT3), Toll-like receptor 2 (TLR2), Toll-like receptor 3 (TLR3), Toll-like receptor 4 (TLR4), Tumor necrosis factor (TNF), and TNF super family member 12 (TNFSF12)
One-way analysis of variance (ANOVA) and Bonferroni multiple comparison correction showed (a) no significant differences in response to filtered air (FA) between males and females in any of the studied groups (SP-A2, co-ex, and KO); (b) no significant differences in response to O3 between males and females in SP-A2 and co-ex, but in contrast, in the absence of surfactant protein A (SP-A) (i.e., KO), a significant difference was observed between sexes (Figure 1A); and (c) significant differences were observed between FA and O3 in KO males (Figure 1A), SP-A2 females (Figure 1B), and in co-ex males and females (Figure 1C)
Summary
Ozone (O3) is a reactive oxidant gas that is a major component of air pollution [1,2]. The proteome profile of AM from the SP-A-KO mice, after treatment with exogenous SP-A1 or SP-A2, resulted in several significant changes in proteins These included proteins involved in the OxS response pathway and actin-related cytoskeletal proteins. Recent studies showed that SP-A1 and SP-A2 differentially regulate in a sex-specific manner the AM [39] and the type II cell [84] miRNome in response to O3 exposure In both cases (AM and type II cell), gonadectomy had a major impact on the miRNome of males when compared to females. SP-A2 (1A0) and co-ex (SP-A1 (6A2)/SP-A2 (1A0)) male and female mice were exposed to filtered air (FA) or O3 and 18 h after exposure the expression level of miRNAs, target mRNAs of the significant miRNAs, and pathways involved were studied.
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